Figure 1.

Initial endocytosis of aptamer HG1-9 mediated by TfR

(A) Representative confocal images of the binding and distribution of aptamer HG1-9 (AF647 conjugated, 50 nM) to TfR-EGFP transfected HeLa cells at 4°C and 37°C for 30 min; scale, 10 μm. (B) The percentage of the uptake of aptamer HG1-9 and hTf by HeLa cells pre-treated with endocytosis inhibitors (50 μM amiloride, genistein, and chlorpromazine [CPZ] and 5 mM methyl-β-cyclodextrin [MβCD]) for 30 min at 37°C; the uptake of HG1-9 by untreated cells was 100%. (C) Flow cytometry analysis of the fluorescence of anti-TfR antibody on the surface of Jurkat cells (representing the content of TfR on cell surface) after incubation with unlabeled hTf (200 nM), HG1-9 (200 nM), and sgc8c (200 nM) at 37°C for a series of incubation times (0, 2, 4, 6, 8 min). The median fluorescence intensity of anti-TfR antibody on cell surface before incubation (0 min) was 100%, and the experiment was conducted three times independently, and each time point has three replicates. (D) Time-resolved spinning disk confocal images of HG1-9 (AF488 conjugated, 50 nM) in Rab5B-mCherry transfected HeLa cells for relative time from 0 s to 136 s; scale, 0.5 μm. (E) Representative confocal images of the binding and distribution of aptamer HG1-9 (AF647 conjugated, 50 nM) to TfR-EGFP and Rab5B-mCherry co-transfected HeLa cells after incubation for 60 min at 37°C; scale, 10 μm. The white arrows indicate the early endosomes containing HG1-9-TfR complex and Rab5B-mCherry near the cell periphery. t test, ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.005; ns, not significant.